Ceabi5 gene, expression vector and application thereof in plant oil regulation

Abstract

The application discloses CeABI5 a gene, an expression vector and application thereof in plant oil regulation. The application first clones a gene with a coding region of a total length of 1125bp, and the protein contains a conserved bZIP domain. CeABI5 A series of vectors of subcellular localization, yeast hybridization and plant overexpression of the gene are constructed. CeABI5 The application first proves that the CeABI5 protein has a transcription activation function in yeast and is located in the cell nucleus, which meets the basic characteristics of a transcription factor. CeABI5 The application proves that the transient overexpression of the gene can significantly improve the oil content of leaves by transforming tobacco through an agrobacterium-mediated microinjection method. CeABI5 The application proves that the overexpression can significantly improve the oil content of seeds and leaves of transgenic Arabidopsis thaliana by transforming the Arabidopsis thaliana through a flower inflorescence dipping method. CeABI5 CeABI5 CeABI5 CeABI5 CeABI5 CeABI5 CeABI5 CeABI The application can be used for improving the oil content of seeds and nutrient tissues of plants.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving CeABI5 Genes, expression vectors and their applications in the regulation of plant oils. Background Technology

[0002] Tiger nuts ( Cyperus esculentus L. var. sativus Boeck, also known as tiger nut, is a perennial herbaceous C4 oilseed crop belonging to the Cyperaceae family of the Poales order. Unlike traditional oilseed crops that accumulate oil in their seeds, tiger nut is the only known crop that accumulates oil at a high level (24%–35%) in its tubers. It is an ideal model for studying the regulation of oil in vegetative tissues, and the discovery and identification of key genes involved in tuber oil accumulation has significant theoretical and applied value.

[0003] ABI5 is a bZIP-type transcription factor, classified under the A subfamily, and is a key downstream transcription factor in the ABA signaling pathway. Extensive research on ABI5 in Arabidopsis thaliana has revealed that it can reduce seed germination rate and has regulatory effects on seed maturation, germination, and seedling growth. ABI5 can also directly target proteins encoding late embryogenesis abundant (LEA). Em1 and Em6 Genes are positively regulated in their expression; Arabidopsis nuclear factor Y family protein (NF-YC9) can directly bind to ABI5, thereby binding to and activating target genes. EM6 The expression of [a specific substance] positively regulates the transcriptional activity of ABI5, thereby participating in the seed germination response to ABA. Studies in wheat have found that... ABI5 The gene expression level increases with increasing wheat seed maturity, indicating its involvement in the regulation of seed development. However, no publicly available data has been published to date. CeABI5 Reports on genes and their role in increasing the oil content of plant seeds and vegetative tissues. Summary of the Invention

[0004] The purpose of this invention is to provide CeABI5 This invention is the first to clone genes, expression vectors, and their applications in plant oil regulation. CeABI5 The gene was used to construct a series of vectors for its subcellular localization, yeast hybridization, and plant overexpression; it was demonstrated for the first time that the CeABI5 protein has transcriptional activation function and is located in the cell nucleus, which is consistent with the basic characteristics of transcription factors; tobacco was transformed using Agrobacterium-mediated microinjection, demonstrating... CeABI5Transient overexpression of the gene significantly increased the oil content in leaves; this was confirmed by transforming Arabidopsis thaliana using the inflorescence immersion method. CeABI5 Gene overexpression can significantly increase the oil content of transgenic seeds and leaves; the cloned and identified genes of this invention... CeABI5 Genes can be used to increase the oil content of plant seeds and vegetative tissues.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides CeABI5 Genes, the ones mentioned CeABI5 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention provides CeABI5 The gene encodes the CeABI5 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] This invention provides a product containing the above-mentioned... CeABI5 The gene expression vector includes one or more of the following: cell localization vector pNC-Cam1304-CeABI5, yeast hybridization vector, and plant overexpression vector pCAMBIA1301-CeABI5; The yeast hybridization vector is one or more of the following: pNC-GBKT7-CeABI5F, pNC-GBKT7-CeABI5N1, pNC-GBKT7-CeABI5N2, pNC-GBKT7-CeABI5C, and pNC-GBKT7-CeABI5TAD.

[0008] Preferably, pNC-Cam1304-CeABI5 is constructed using primers CeABI5F2 and CeABI5R2; Alternatively, pCAMBIA1301-CeABI5 can be constructed using primers CeABI5F5 and CeABI5R5; pNC-GBKT7-CeABI5F was constructed using primers CeABI5F2 and CeABI5R2; pNC-GBKT7-CeABI5N1 was constructed using primers CeABI5F2 and CeABI5R3; pNC-GBKT7-CeABI5N2 was constructed using primers CeABI5F2 and CeABI5R4; pNC-GBKT7-CeABI5C was constructed using primers CeABI5F3 and CeABI5R2; pNC-GBKT7-CeABI5TAD was constructed using primers CeABI5F4 and CeABI5R3; The nucleotide sequence of CeABI5F2 is shown in SEQ ID NO.5; The nucleotide sequence of CeABI5R2 is shown in SEQ ID NO.6; The nucleotide sequence of CeABI5F3 is shown in SEQ ID NO.7; The nucleotide sequence of CeABI5R3 is shown in SEQ ID NO.8; The nucleotide sequence of CeABI5F4 is shown in SEQ ID NO.9; The nucleotide sequence of CeABI5R4 is shown in SEQ ID NO.10; The nucleotide sequence of CeABI5F5 is shown in SEQ ID NO.11; The nucleotide sequence of CeABI5R5 is shown in SEQ ID NO.12.

[0009] This invention provides a method for amplifying the above CeABI5 Primer pairs for the gene, said primer pairs including CeABI5F1 and CeABI5R1; The nucleotide sequence of CeABI5F1 is shown in SEQ ID NO.3; The nucleotide sequence of CeABI5R1 is shown in SEQ ID NO.4.

[0010] This invention provides the aforementioned CeABI5 The application of the gene, the CeABI5 protein, the expression vector, or the primer pair in the regulation of plant oils; The plants include one or more of the following: Arabidopsis thaliana, tobacco, rapeseed, soybean, peanut, sunflower, mustard greens, cabbage, tomato, potato, sweet potato, cassava, beet, corn, wheat, sorghum, sugarcane, oil palm, and coconut.

[0011] Preferably, the regulation of plant oils includes: increasing the concentration of plant oils in the plant oil content of oils. CeABI5 The level of gene expression promotes the accumulation of oil in plant seeds or plant tissues, thereby increasing the oil content.

[0012] Preferably, the plant tissue includes one or more of the following: leaves, stems, tubers, and rhizomes.

[0013] This invention provides a method for increasing the oil content of plants or promoting the accumulation of plant oils, comprising the following steps: The above CeABI5 Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants.

[0014] Preferably, the target plant includes one or more of Arabidopsis thaliana and tobacco.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention was first obtained and disclosed. CeABI5 The gene sequence has a coding region of 1125 bp in length; (2) This invention has constructed a series of vectors for gene subcellular localization, yeast hybridization, and plant overexpression, and has for the first time demonstrated that... CeABI5 The encoded protein has transcriptional activation function and is located in the cell nucleus, which conforms to the basic characteristics of transcription factors; (3) This invention proves CeABI5 Transient overexpression of the gene in tobacco can significantly increase the oil content of leaves. CeABI5 Overexpression of the gene in Arabidopsis thaliana significantly increased the oil content of transgenic seeds and leaves, demonstrating... CeABI5 It has the function of promoting oil accumulation and shows its application prospect in increasing the oil content of seeds and nutrient tissues. Attached Figure Description

[0016] Figure 1 For the present invention CeABI5 PCR amplification results of the gene: where M: DNA marker III; CK: blank control; 1: CeABI5 ; Figure 2 For the present invention CeABI5 BLASTN alignment of genes in the NCBI GenBank database; Figure 3 This is a schematic diagram of the sequence characteristics and evolutionary analysis of CeABI5 in this invention; where A: analysis of conserved domains of CeABI5; B: sequence alignment of CeABI5 and AtABI5; C: evolutionary analysis of CeABI5 and homologous proteins in rice, maize, Arabidopsis thaliana, wheat and barley; Figure 4 This is a subcellular localization result of the CeABI5 protein in tobacco leaf cells according to the present invention; where the bright field is the image taken under conditions without fluorescence to capture the complete cell outline and cell morphology, and the superimposed field is the superimposed imaging effect of green fluorescence signal + red fluorescence signal + bright field. Figure 5 The figure shows the results of identifying the transcriptional activation function of CeABI5 in yeast according to the present invention: SD / T is a tryptophan-deficient medium, SD / TH is a tryptophan and histidine-deficient medium, and SD / TH+X-α-gal is a tryptophan and histidine-deficient medium supplemented with X-α-gal (5-bromo-4-chloro-3-indole-α-D-galactopyranoside). Figure 6For the present invention CeABI5 Transient overexpression of the gene in tobacco leaves and its regulatory effect on lipid accumulation: The horizontal axis represents different days after transformation, and the vertical axis represents the experimental group (…). CeABI5 The fold increase in triglycerides (TAG) in the control group (transformed with empty vector WT) and the control group is shown as the average of three biological replicates. Lowercase letters indicate the basis of measurement. P The difference was statistically significant (<0.05). Figure 7 For the present invention CeABI5 Results of the regulatory effect of gene overexpression on seed and leaf lipid accumulation in Arabidopsis thaliana; Figure A: Experimental group ( CeABI5 B: Fold increase in TAG in T3 generation seeds of transgenic plants relative to empty vector T3 generation plants; (A) Fold increase in TAG in leaves of transgenic plants relative to empty vector T3 generation plants, showing the average of 3 biological replicates, "**" indicates based on... P The difference was statistically significant (<0.01). Detailed Implementation

[0017] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention. The method for constructing the expression vector described in this invention has no special requirements and can be constructed using methods known in the art.

[0018] In this invention, the primers used are shown in Table 1.

[0019] Table 1 CeABI5 Primers used for gene cloning and vector construction

[0020] The present invention CeABI5 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the specific sequence information is as follows:

[0021] The present invention CeABI5 Gene-encoded CeABI5 The amino acid sequence of the protein is shown in SEQ ID NO.2, and the specific sequence information is as follows: MASPVSEDTEVTSQHRSPHLHAAALDQAQAPAQGQGQYSAEKDFGYPLAHQNSIYSLTLDEIQNTVCEPGRNFGSMNMDEFLTNIWNAEEGQALGQATTSASASLAGAADVVVAPALQRQASLTVPPPLSRKTVEEVWTEIHHRASQGGPVTPPGAQPAVNGGMAAANGSRQPTFGEMTLEDFLIKAG VVREPTVSVRVAPPQFGQMPVYPMGVDMGAAGMYGAPMMGGPNMNLNMNGGMMHGMMVGPVGSPGTPDGMGDDVAGYDRVRRKRSAGAAGMGGEGMVVEKGVERRQRRMIKNRESAARSRARKQAYTVELEAELNQLKEENARLKEEEKKVLQLKRQMLLEAITEQAYVHAQQAKGSLRRSFTTCTW*.

[0022] Example 1 CeABI5 Gene cloning and sequence analysis (1) Using tubers of Tiger Pea No. 3 at different developmental stages as material, total RNA was extracted using the Tiangen Plant Polysaccharide Polyphenol RNA Extraction Kit (catalog number DP441): (2) The above mRNA was reverse transcribed into cDNA using the Takara PrimeScript™ RT reagent Kit with gDNA Eraser, which was used as a template for PCR amplification; (3) Based on the full-length cDNA obtained from the transcriptome, primer pairs CeABI5F1 (SEQ ID NO.3) and CeABI5R1 (SEQ ID NO.4) were designed using Primer Premier 5.0 as shown in Table 1. (4) After optimizing the PCR conditions according to the Tm values ​​of the primers, PCR amplification was performed using CeABI5F1 (SEQ ID NO.3) and CeABI5R1 (SEQ ID NO.4) as primers and the cDNA obtained by reverse transcription as a template. The first round of PCR amplification reaction system is shown in Table 2. The PCR reaction program is as follows: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 60 sec (35 cycles); 72℃ for 5 min; stored at 4℃. Table 2 First-round PCR amplification reaction system

[0023] (5) PCR amplification yielded a specific band of approximately 1000 bp. Figure 1 After being recovered from the gel using the OMEGA gel recovery kit, the cells were cloned into the pMD19-T cloning vector from Takara. The sequence was determined after blue-white screening and colony PCR verification. (6) Sequence analysis showed that, CeABI5 The coding region (CDS) is 1125 bp in length, with a GC content of 45.65%, and is predicted to encode 374 amino acids. Its theoretical molecular weight is 40.18 kDa, isoelectric point (pI) is 6.28, total average hydrophobicity index (GRAVY) is -0.477, instability coefficient (II) is 52.44, and aliphatic index (AI) is 65.03.

[0024] (7) BLASTN alignment using the NCBI GenBank database did not reveal any similar sequences. Figure 2 This indicates that it is a new gene; (8) CDD (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?) analysis and alignment with the AtABI5 sequence showed that CeABI5 contains one conserved bZIP domain ( Figure 3 In the A), activation domain (TAD) ( Figure 3 (B in the middle) (9) To further reveal the evolutionary characteristics of CeABI5, a phylogenetic tree was constructed using MEGA6 (https: / / www.megasoftware.net / ) with ABI5 proteins from Arabidopsis thaliana (AtABI5), maize (ZmABI5), rice (OsABI5), wheat (TaABI5), and barley (HvABI5). Figure 3 As shown in C, CeABI5 clusters with OsABI5 and TaABI5, with sequence identity of 45.3% and 41.5%, respectively.

[0025] Example 2 Subcellular localization analysis of CeABI5 protein (1) WoLF PSORT (https: / / www.genscript.com / wolf-psort.html) analysis showed that CeABI5 protein may be located in the cell nucleus; (2) To confirm the above prediction results, primer pairs CeABI5F2 (SEQ ID NO.5) and CeABI5R2 (SEQ ID NO.6) as shown in Table 1 were designed near the start and stop codons of the sequencing gene. (3) Using the PCR product diluted 100 times in Example 1 as a template, CeABI5F2 (SEQ ID NO.5) and CeABI5R2 (SEQ ID NO.6) as primers, PCR amplification was performed. The reaction system for the second round of PCR amplification is shown in Table 3. The PCR reaction program was: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 60 sec (35 cycles); 72℃ for 5 min; and stored at 4℃. After gel extraction and recovery, the PCR product was cloned into pNC-Cam1304-SubN using the NC kit (purchased from Hainan Nixing Biotechnology Co., Ltd.) to construct the fusion expression vector pNC-Cam1304-CeABI5 with EGFP. Table 3. Second-round PCR amplification reaction system

[0026] Table 4 NC Connection System

[0027] (4) Transform the recombinant plasmid pNC-Cam1304-CeABI5 into Agrobacterium strain GV3101 (containing pSoup-P19), the specific steps are as follows: (4-1) Add 1 μg of empty vector plasmid pNC-Cam1304-SubN and 1 μg of recombinant plasmid pNC-Cam1304-CeABI5 to 100 μL of competent cells GV3101 and mix well by pipetting. (4-2) Ice bath for 30 min, then quickly place in liquid nitrogen for 1 min, and then heat shock in a water bath at 37℃ for 5 min; (4-3) Add 950 μL of antibiotic-free liquid YEP medium and incubate at 28℃ and 200 rpm for 4 h with shaking. (4-4) Centrifuge at 10,000 rpm for 1 min to concentrate the bacterial culture, discard the supernatant and resuspend the bacterial cells in 100 μL of antibiotic-free liquid YEP medium; (4-5) Spread the bacterial cells on solid YEP medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampin, and incubate at 28°C for 2-3 days; (4-6) Select single clones for colony PCR detection and screen positive clones for subsequent genetic transformation experiments.

[0028] (5) The leaves of 4-week-old tobacco plants were transformed using the micro-injection method. The specific steps are as follows: (5-1) Inoculate the above-mentioned positive bacteria into 2 mL of LB liquid medium (50 mg / L rifampin) and incubate overnight at 28°C and 210 rpm; (5-2) Take 1 mL of bacterial culture and add it to 30 mL of LB liquid medium. Incubate at 28°C and 210 rpm until the bacterial concentration reaches OD500. 600 It is 0.8; (5-3) Collect bacterial cells by centrifugation at 5000 rpm for 3 min, remove the supernatant, and add resuspension buffer (10 mmol / L MgCl2, 0.2 mmol / L acetylsalicylic acid and 10 mmol / L MES, adjust pH to 5.6 using KOH) to resuspend the bacterial cells. Repeat twice, adjusting the concentration of the resuspension to OD. 600 It is 0.6; (5-4) After resuspending the bacterial solution, incubate it at 28°C for 3-5 hours. Then, inject it into the lower epidermis of the tobacco plant using a 1mL syringe with the needle removed (one gene is injected into multiple leaves of one tobacco plant). You can use the needle to slightly puncture the lower epidermis of the leaf before injection. (5-5) After the tobacco leaves were cultured in the dark for 2-4 days after injection, the injected tobacco leaves were taken to prepare slides for fluorescence observation. (6) Using a laser confocal microscope, the green fluorescent signal (GFP) showed the location of the CeABI5 protein, and the red fluorescent signal (Red) showed the location of the cell nucleus. The superposition field showed an orange-yellow color, indicating that the green fluorescent signal in the experimental group and the red fluorescent signal labeled in the cell nucleus highly overlapped, indicating that the CeABI5 protein plays a role in the cell nucleus. Figure 4 ).

[0029] Example 3 Identification of the transcriptional activation function of CeABI5 protein (1) To identify the transcriptional activation function of CeABI5 protein and the location of the activation domain, primers as shown in Table 1 were designed near the bZIP domain. (2) Using the PCR product diluted 100 times in Example 1 as a template, CeABI5F2 (SEQ ID NO.5) and CeABI5R2 (SEQ ID NO.6) were used as primers to amplify the full length of the coding region of CeABI5 (SEQ ID NO.1). After the PCR amplification was completed, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI5F; Using the PCR product diluted 100-fold in Example 1 as a template, the N1 end (i.e., the first 1-868 bp of the nucleotide sequence shown in SEQ ID NO.1, upstream of the bZIP domain) was amplified using CeABI5F2 (SEQ ID NO.5) and CeABI5R3 (SEQ ID NO.8) as primers. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI5N1. Using the PCR product diluted 100-fold in Example 1 as a template, the N2 end (i.e., the 1st to 576th bp of the nucleotide sequence shown in SEQ ID NO.1, containing the transcription activation domain) was amplified using CeABI5F2 (SEQ ID NO.5) and CeABI5R4 (SEQ ID NO.10) as primers, respectively. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI5N2. Using the PCR product diluted 100-fold in Example 1 as a template, the TAD end (i.e., the 178-576 bp of the nucleotide sequence shown in SEQ ID NO.1, the transcription activation domain) was amplified using CeABI5F4 (SEQ ID NO.9) and CeABI5R3 (SEQ ID NO.8) as primers. After PCR amplification, the product was excised from the gel and cloned into pNC-GBKT7 using an NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI5TAD. Using the PCR product diluted 100-fold in Example 1 as a template, the C-terminus (i.e., the 869-1125 bp of the nucleotide sequence shown in SEQ ID NO.1, containing the bZIP domain) was amplified using CeABI5F3 (SEQ ID NO.7) and CeABI5R2 (SEQ ID NO.6) as primers. After PCR amplification, the product was excised from the gel and cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI5C. The PCR amplification reaction system, reaction procedure, and NC ligation system described above are the same as those in Example 2. (3) The empty vector (pNC-GBKT7) and 5 recombinant plasmids were transferred into Y190 yeast cells. Then, the successfully transformed yeast cells were screened on tryptophan-deficient medium (SD / T, purchased from Beijing Cooler Technology Co., Ltd.). Positive bacteria were then selected and inoculated on tryptophan and histidine-deficient medium (SD / TH, purchased from Beijing Cooler Technology Co., Ltd.) for transcriptional autoactivation activity screening. Subsequently, a colorimetric reaction was performed on tryptophan and histidine-deficient medium (SD / TH+X-α-gal) supplemented with X-α-gal (5-bromo-4-chloro-3-indole-α-D-pyranogalactoside).

[0030] (4) The results showed that the engineered bacteria transformed with pNC-GBKT7-CeABI5F, pNC-GBKT7-CeABI5N1, pNC-GBKT7-CeABI5N2, and pNC-GBKT7-CeABI5TAD could survive on tryptophan- and histidine-deficient media and showed a blue color, while yeast cells transformed with empty vector and pNC-GBKT7-CeABI5C failed to show β-galactosidase activity. This indicates that the CeABI5 protein has transcriptional activation function, and its activation domain is located upstream of the bZIP domain, i.e., positions 59-192 of the protein, corresponding to positions 178-576 of the nucleotide sequence (…). Figure 5 ).

[0031] Example 4 CeABI5 Identification of gene regulation of lipids in tobacco (1) For identification CeABI5 The function of the gene in the regulation of plant oils was investigated by using the PCR product diluted 100 times in Example 1 as a template and CeABI5F5 (SEQ ID NO.11) and CeABI5R5 (SEQ ID NO.12) as primers for PCR amplification. After the target fragment was recovered from the gel, it was cloned into the plant overexpression vector pCAMBIA1301 using homologous recombination to construct the recombinant vector pCAMBIA1301-CeABI5. (2) The plant overexpression vector pCAMBIA1301-CeABI5 constructed above was transformed into Agrobacterium strain GV3101 (containing pSoup-P19). (3) The above-mentioned Agrobacterium tumefaciens containing the empty vector and the plant overexpression vector pCAMBIA1301-CeABI5 was used to transform 4-week-old tobacco leaves by micro-injection. (4) Samples were collected 1 day, 3 days and 5 days after transformation for gene expression and lipid content determination; (5) Oil content determination results based on Agilent 7890A (HP-FFAP, 30m×0.25mm ID, 0.25μm, Santa Clara, CA, USA) showed that, compared with the control group transformed with empty vector, the TAG content in the experimental group increased by 2.2 times, 2.9 times, and 2.6 times after 1 day, 3 days, and 5 days of transformation, respectively, with a significant difference between 3 days and 1 day. Figure 6 ).

[0032] Example 5 CeABI5 Application of genes in the regulation of lipids in Arabidopsis seeds and vegetative tissues (1) For evaluation CeABI5 To explore the potential of genes in plant oil regulation, wild-type Arabidopsis thaliana was transformed using the inflorescence immersion method with Agrobacterium tumefaciens engineered strains containing empty vectors and pCAMBIA1301-CeABI5, respectively. The specific steps of Arabidopsis thaliana transformation are as follows: (1-1) Positive bacteria were cultured overnight in LB liquid containing 50 mg / L kanamycin and 100 mg / L rifampin until OD reached. 600 The bacterial cells were collected by centrifugation at 12,000 rpm for 5 minutes with a concentration of 0.6. (1-2) Resuspend in staining buffer (1 / 2 MS solution containing 5% sucrose and 0.02% Silwet-77) and adjust to OD. 600 It is 0.8; (1-3) Place the Arabidopsis thaliana upside down in the infusion solution and soak for half a minute. Try to avoid getting the leaves in the infusion solution. You can use a pipette to draw the solution and then drip it onto the inflorescence. (1-4) After the inoculation is completed, spray a small amount of water and cover the inflorescence with a black plastic bag for dark culture. After 1 day, remove the plastic bag and culture normally. (1-5) To improve the efficiency of the infiltration, the infiltration can be repeated one or two times (once a week), and the seeds can be cultivated under the same conditions until they mature and are harvested. (1-6) T1 seeds were screened for resistance on MS solid plate medium with 30 µg / mL hygromycin to obtain T2 generation positive transgenic plants. After they matured, they were screened for resistance again to obtain stable T3 generation positive transgenic plants.

[0033] (2) To analyze the effect of gene overexpression on seed oil regulation, oil content was measured in T3 generation transgenic Arabidopsis seeds. The results showed that, compared with the control group (WT) containing the empty vector, CeABI5 The accumulation of TAGs in overexpressed seeds increased significantly, by approximately 37.4%. Figure 7 (A in the middle) (3) For evaluation CeABI5To investigate the application value of oil regulation in plant vegetative tissues, leaves of the aforementioned T3 generation transgenic Arabidopsis thaliana were collected for oil content determination. The results showed that, compared with the control group (WT) containing empty vector, oil content was significantly higher. CeABI5 The oil content of the leaves increased by 2.42 times due to overexpression. Figure 7 (B in the middle).

[0034] (4) The above results indicate that, CeABI5 It has the function of promoting oil accumulation and shows its application prospect in increasing the oil content of seeds and nutrient tissues.

[0035] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kind CeABI5 Genes or containing CeABI5 The application of plant overexpression vectors for gene regulation in Arabidopsis or tobacco oil is characterized by, The CeABI5 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The oil regulation includes: increasing the levels of oil in Arabidopsis or tobacco. CeABI5 The level of gene expression promotes the accumulation of oil in the seeds or leaves of Arabidopsis or tobacco, thereby increasing the oil content.

2. The application according to claim 1, characterized in that, contain CeABI5 The plant overexpression vector for the gene is pCAMBIA1301-CeABI5.

3. A method for increasing the oil content of plants or promoting the accumulation of plant oils, characterized in that, Includes the following steps: The claim 1 CeABI5 Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants. The target plant is one or more of Arabidopsis thaliana and tobacco.

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